New insights from age determination on toxic element accumulation in striped and bottlenose dolphins from Atlantic and Mediterranean waters

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New insights from age determination on toxic element accumulation in striped and bottlenose dolphins from

Atlantic and Mediterranean waters

Virginie Lahaye, Paco Bustamante, Willy Dabin, Olivier van Canneyt, Franck Dhermain, C. Cesarini, Graham Pierce, Florence Caurant

To cite this version:

Virginie Lahaye, Paco Bustamante, Willy Dabin, Olivier van Canneyt, Franck Dhermain, et al.. New insights from age determination on toxic element accumulation in striped and bottlenose dolphins from Atlantic and Mediterranean waters. Marine Pollution Bulletin, Elsevier, 2006, 52 (10), pp.1219-1230.

�10.1016/j.marpolbul.2006.02.020�. �hal-00226358�

New insights from age determination on toxic element accumulation in striped and bottlenose dolphins from Atlantic and Mediterranean waters

V. Lahaye

, P. Bustamante

, W. Dabin

, O. Van Canneyt

, F. Dhermain

, C. Cesarini

, G.J. Pierce

, F. Caurant

a

Laboratoire de Biologie et Environnement Marins, FRE 2727 du CNRS, Université de La Rochelle, 22 Av. Michel Crépeau, 17042 La Rochelle Cedex, France

b

Centre de Recherche sur les Mammifères Marins, Institut du Littoral et de l’Environnement, Port des Minimes, Av. du Lazaret, 17000 La Rochelle, France

c

Groupe d’Etude des Cétacés de Méditerranée, Clinique Vétérinaire du Redon, 13 Bd du Redon, 13009 Marseille, France

d

Departement of Zoology, School of Biological Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen, ABZ4 2TZ, UK

*

Corresponding author. Tel./Fax.: +33 (0)5 46 50 02 94.

E-mail address: vlahaye@univ-lr.fr (V. Lahaye).

Abstract

This study aimed at comparing toxic element (Hg, Cd) bioaccumulation in relation to age for bottlenose (Tursiops truncatus) and striped dolphins (Stenella coeruleoalba) from Mediterranean and Atlantic waters. Metal concentrations were also measured in selected prey to infer metal exposure through the diet. As expected, Mediterranean prey exhibited the highest Hg levels, probably as a consequence of the Hg enrichment of the Mediterranean Sea.

Comparing the predators from each area and taking age into account, Mediterranean bottlenose dolphins displayed higher Hg levels than Atlantic dolphins (p = 0.032), whereas Mediterranean striped dolphins did not (p = 0.691). The consumption of Myctophid fish, which showed the highest Hg levels (105 ± 80 ng.g

w.wt.) among Atlantic prey, may explain the high Hg levels in the liver of the Atlantic striped dolphins and suggested a preferential offshore feeding behaviour in this area. Concerning Cd, no clear differences were found between geographical areas.

Keywords: Stenella coeruleoalba; Tursiops truncatus; Mercury; Cadmium; Diet; Exposure

1. Introduction

Due to their terminal position in the food web and their long life span, carnivorous marine mammals accumulate particularly high levels of mercury (Hg) and cadmium (Cd) (Wagemann and Muir, 1984; Aguilar et al., 1999). The natural occurrence of these trace elements in seawater (Nriagu, 1996) has involved adaptations of marine organisms over geological time scales to the presence of toxic metals in their environment. Thus, marine mammals have developed efficient detoxification capacities to support elevated exposure to these metals (see reviews by Cuvin-Aralar and Furness, 1991 and Das et al., 2000a). The demethylation of organic Hg in the liver leads to the production of non-toxic granules of tiemannite (Koeman et al., 1973; Martoja and Berry, 1980). The accumulation of tiemannite granules makes the liver the ultimate organ of retention of Hg (Wagemann et al., 1998; 2000).

Since these granules are not excreted (Nigro and Leonzio, 1996), inorganic Hg would be

stored in this organ for nearly the whole life. This detoxification process results in particularly

elevated Hg concentrations in the liver, which is not the case when considering levels in the

kidneys, where no storage of Hg under tiemannite would occur (Cuvin-Aralar and Furness,

1991). Like Hg in the liver, Cd levels can also be particularly elevated in the kidneys of

marine mammals (Wagemann and Muir, 1984; Dietz et al., 1998). Its potential toxic effects

are mitigated by the binding to metallothioneins both in the liver and the kidneys (Klaassen

and Liu, 1997; Teigen et al., 1999). In other mammals such as humans, the biological half-life

of Cd has been estimated to be a few months in liver and between 10 to 30 years in kidneys

(Friberg et al., 1974), and Cd could also be stored over very long time in the renal tissue of

marine mammals.

In addition, Cd and Hg concentrations display an important variability in marine mammal tissues, which may be the result of biological and ecological influences (Aguilar et al., 1999).

At the individual scale, the most important parameter to be considered is the age, since these metals potentially accumulate throughout the life. Other factors such as growth features, gender, reproductive status and/or health aspects may also be important. At the population scale, feeding preference is probably the key factor controlling Cd and Hg levels because upper-level predators are mainly exposed to metals through their food (Aguilar et al., 1999).

Feeding on cephalopods is probably a major source of Cd for small cetaceans both because of the high Cd levels in these prey and because Cd is present mainly in bioavailable forms in their tissues (Bustamante et al., 1998; 2002). In both fish and cephalopods, Hg occurs mainly in organic forms which are highly bioavailable for the predators (Bloom, 1992; Bustamante et al., submitted). As a result, species or populations that consume an important proportion of cephalopods can be expected to exhibit higher Cd levels in their tissues than piscivorous upper-level predators. The effects of feeding preferences on metal exposure may also be combined with other characteristics inherent to the species, the habitat and the geographical location, as concentrations in prey would reflect concentrations in their environment (e.g.

Law et al., 1991; 1992).

The present study aims at determining the main biological and ecological factors influencing Cd and Hg concentrations in two small cetacean species inhabiting the French waters, the striped dolphins (Stenella coeruleoalba) and the bottlenose dolphins (Tursiops truncatus) (e.g.

Forcada et al., 1990; Goujon et al., 1993; Gannier, 1995; Liret et al., 1998). The two

geographical areas involved are the Mediterranean Sea and the Bay of Biscay. These areas are

characterised by different oceanological features which are likely to affect metal exposure to

small cetaceans. Indeed, the Mediterranean Sea is well-known for its natural Hg enrichment

(e.g. Bacci, 1989; Cossa et al., 1997). Furthermore, a previous study has reported that striped dolphins from the Mediterranean Sea exhibited much higher Hg levels than those from the Atlantic (André et al., 1991). However, this report did not take age into account, only body length, which may introduce a bias. Indeed, the use of length as a covariate to detect differences in pollutant levels between areas does not permit comparisons between individuals having reached their asymptotic length or/and between groups with different growth curves (Monaci et al., 1998). Striped dolphins from the Mediterranean Sea are smaller than those from the Atlantic (Di-Méglio et al., 1996), which may affect Hg levels in these two populations.

As a consequence, understanding accumulation of toxic elements with age is mandatory to establish the effective metal impregnation state of small cetaceans from the Mediterranean and Atlantic waters and to allow further comparison between both areas. Renal Cd and hepatic Hg are measured in order to provide a long-term overview of the impregnation. In addition, the influence of diet on metal exposure to striped and bottlenose dolphins is investigated using metal concentrations in reported prey from each area. Thus, the influence of diet on metal levels in these small cetaceans could be clarified both at the intra- and inter- specific scale.

2. Material and methods

2.1. Studied areas

The Bay of Biscay is situated in the north-eastern Atlantic and extends from 1 to 10°W and 43

to 48°N (Fig. 1). This area is characterised by a variable distance of the shelf-edge from the

coastline, the continental shelf width varying from 60 to 100 nautical miles (NM) in the northern part of the Bay (up to 45°N) to 25-30 NM in the southern part, and reaching 3 NM at the latitude of the Capbreton trough. Within this area, Arcachon Bay (see Fig. 1) constituted a semi-enclosed shallow habitat for bottlenose dolphins until 2001, when the last individual of this small resident population was found stranded (Ferrey et al., 1993; J.J. Boubert, pers.

comm.).

In contrast, the continental shelf width is very limited along the French Mediterranean coasts, with the largest width encountered in the Gulf of the Lion (15 to 45 NM, see Fig. 1), as compared to 2.5-10 NM at the eastern of 5°30 E. These latter areas are characterised by an abrupt shelf-edge, with deep canyons (up to - 2000m) close to the coastline.

2.2. Biological material

2.2.1. Small cetaceans

Between 1999 and 2004, 55 stranded small cetaceans were sampled along the whole French

Mediterranean (n = 15) and Atlantic coasts (n = 40) by the Réseau National Echouage (RNE)

co-ordinated by the Centre de Recherche sur les Mammifères Marins (CRMM) of La

Rochelle (France). Striped dolphins were the most common species (n = 40) whereas only 15

bottlenose dolphins were collected. Among the 10 Atlantic bottlenose dolphins, 3 animals

were identified as the last bottlenose dolphins from the small resident group of Arcachon Bay

(J.J. Boubert, pers. comm.), the history of which is well-known (Ferrey et al., 1993). During

the necropsies, teeth (n = 55) were collected for age determination, and liver (n = 55) and

kidney (n = 48) for trace element analyses (Table 1). These samples were stored at –20°C

until being processed in the laboratory.

Age was determined at the CRMM, following the recommendations of Perrin and Myrick (1980). Briefly, this procedure consists of counting Growth Layer Groups (GLGs) from teeth sections, assuming that one GLG equals one year. All carcasses were in good post-mortem condition, except a single striped dolphin from the Mediterranean Sea. Owing to the small number of dolphins collected along the Mediterranean coast (see Table 1), the single putrefied animal has been included in the study. This individual can be identified in figures since it is the oldest striped dolphins (i.e. 27 years-old).

2.2.2. Prey

Selected prey from each area were also sampled for toxic element analyses. The selection of species and their size was based upon previous dietary studies on bottlenose and striped dolphins originating from the study areas (Würz et al., 1992; Blanco et al., 2001; Spitz, 2004;

Astruc et al., in press; Pusineri et al., in press; Spitz et al., submitted). Prey sampling occurred during IFREMER groundfish surveys, in winter and spring 1997 and in autumn 2001-2003 (RESSGASC and EVHOE campaigns, respectively). For the Mediterranean Sea, sampling was conducted in May and October 2004 (MERMED campaigns). Whereas RESSGASC and MERMED surveys occurred only on the continental shelf, EVHOE surveys extended to the shelf-edge, allowing the collection of mesopelagic species. All prey samples were immediately frozen at -20°C on board and then stored until being processed in the laboratory.

After species determination, each prey was weighed and measured. Metal analyses were

carried out using the whole prey in order to reflect the exposure to predators. As cephalopods

represent a major pathway of Cd exposure (Bustamante et al., 1998; 2002) and since Cd

levels are particularly low in fish (Lahaye et al., in press), Cd levels were assayed in cephalopods but not in fish. In contrast, the presence of Hg in its bioavailable form in both fish and cephalopod (Bloom, 1992; Bustamante et al., submitted) necessitates measurements of Hg concentrations in both prey items.

2.3. Metal analyses

All equipment used in the sample processing was cleaned, and subsequently decontaminated for 24 h in a solution composed of 35 ml HNO

(65%) and 50 ml HCl (36%) for 1 L of Milli- Ro quality water. Fresh samples were freeze-dried and ground to powder. The mean ratio between dry weight (d.wt.) and wet weight (w.wt.) was 0.28 for liver and 0.23 for kidney.

Each sample was then treated in duplicate.

For total Hg measurements, aliquots ranging from 0.2 to 2 mg of dried-material were analysed in an Advanced Mercury Analyser spectrophotometer (Altec AMA 254). Hg determination in the AMA 254 involved evaporation of Hg by progressive heating to 800°C under oxygen atmosphere for 3 min, and subsequent amalgamation on a gold net. The net was then heated to liberate the collected Hg, which was measured by UV atomic-absorption spectrophotometry.

For Cd analyses, 2 aliquots of approximately 200 mg of each homogenised dry sample were

digested with 3.5 ml of 65% HNO

at 60°C for 3 days. The digested contents were then

diluted to 10 ml in milli-Q quality water. Then Cd contents were assayed using a flame

(Varian 250 Plus) and graphite furnace (Hitachi Z-5000) atomic absorption spectrophotometer

with deuterium and Zeeman background correction, respectively.

Quality controls were made using standard reference materials (National Research Council of Canada). These standards were treated and analysed under the same conditions as the samples and results were in good agreement with the certified values (Table 2). In addition, intercalibration exercices were carried out on material from the International Atomic Energy Agency. Detection limits were determined with blank analyses and the obtained values were 0.005 µg.g

d.wt. for Hg, 0.4 and 0.002 µg.g

d.wt. for Cd, respectively using flame and furnace AAS.

2.4. Data treatment

Metal concentrations were expressed as µ g.g

wet weight (w.wt.). All concentrations below

the detection limit were replaced with “dummy values” that were half of the detection limit in

order to allow further statistical comparisons (Gibbons and Coleman, 2001). Figures showing

metal concentrations against age were presented on a logarithmic scale in order to better

describe the relationship of metals with age in the youngest dolphins since they constituted a

large part of the sampling (see Table 1). Statistical analyses were performed using XL-STAT

Pro 7.0. Metal concentrations were natural log-transformed in order to reduce skewness when

striped dolphins were taken into account. When the relationship of metal concentrations with

age was linear, analysis of covariance (ANCOVA, Quinn & Keough 2002) was used to

determine the influence of gender and area, with age as the covariate. When no influence of

age on metal concentrations was established, analyse of variance (ANOVA) was used to

compare groups (i.e. per gender or area).

3. Results

3.1. Metal levels in bottlenose dolphins

Concentrations of both metals displayed high variability: overall coefficients of variations (CV) were 96% for Hg in liver and 103% for Cd in kidneys. Hepatic Hg concentrations ranged from 1.5 to 331 µg

w.wt. (Fig. 2a) and renal Cd concentrations ranged from 0.06 to 3.05 µg.g

w.wt. (Fig. 2b).

Correlation with age was significant for both hepatic Hg (p = 0.001) and renal Cd (p < 0.0001, Table 3). As the three bottlenose dolphins from Arcachon Bay constituted a particular social unit, these individuals were not taken into account for the global geographical comparison (Mediterranean Sea vs Atlantic Ocean). Figure 2 shows that Mediterranean bottlenose dolphins exhibited much higher Hg levels in liver than Atlantic ones (p = 0.032, Table 3) whereas Cd levels were rather similar in these two groups (p = 0.207, Table 3).

The three 3 individuals from Arcachon Bay exhibited very low Cd levels compared to the other Atlantic animals (Fig. 2b). Among the Arcachon Bay animals, two individuals exhibited hepatic Hg concentrations as high as those of Mediterranean bottlenose dolphins (Fig. 2a).

3.2. Metal levels in striped dolphins

The global CV of hepatic Hg was particularly elevated for striped dolphins (i.e. 221%). The highest concentration was found in a 27-year-old Mediterranean striped dolphin (1033 µg.g

w.wt; see Fig. 3a), which was also the single putrefied specimen of the overall sampling.

Concentration variability was much lower for renal Cd, with a CV of 94%. Surprisingly, the

maximal value of renal Cd was obtained in a relatively young Atlantic striped dolphin (age estimated to be 1.3-year-old) reaching 40.2 µ g Cd.g

w.wt. (see Fig. 3b).

Metal accumulation with age in striped dolphins was only apparent for hepatic Hg (Fig. 3a; p

< 0.0001, Table 3). In addition, no influence of the geographical area on the bioaccumulation rates was observed for this metal (p = 0.691, Table 3). In contrast, renal Cd concentrations would increase until 2 years-old, and then would remain independent of the age (Fig. 3b).

However, when looking at calves of 2 years-old and less, Cd concentrations in the kidneys were more influenced by the geographical area (p = 0.018) than the age (p = 0.635) and the interaction between these two parameters was not significant (p = 0.741, Table 3). In addition, although the Mediterranean sampling was limited compared to the Atlantic sampling, the two adult Mediterranean striped dolphins exhibited quite similar renal Cd concentrations to the Atlantic ones (Fig. 3b).

The high number of striped dolphins sampled along the Atlantic coast (n = 30; see Table 1) allowed us to consider the influence of gender on metal accumulation. No influence of gender on metal concentrations was established for either hepatic Hg (p = 0.637) or renal Cd (p = 0.515, Table 4).

3.3. Inter-specific comparisons

Owing to the limited Mediterranean sampling, inter-specific comparisons were carried out

only for the Atlantic. Since bottlenose dolphins from Arcachon Bay had lived isolated for a

long time and therefore displayed different metal concentrations compared to the other

individuals from the Atlantic coast, they were not included in the inter-specific comparison.

Bioaccumulation rates of Hg in liver with age were similar in Atlantic bottlenose and striped dolphins (p = 0.774) whereas Cd bioaccumulation in the kidneys with age was significantly higher in striped dolphins than in bottlenose dolphins (p = 0.026, Table 5).

3.4. Metal levels in prey

Mediterranean prey displayed higher Hg levels than Atlantic ones, the mean length of the prey analysed being equivalent for both areas. In addition, cephalopod and fish exhibited similar Hg concentrations, the highest level being reached in a cephalopod specimen, Illex coindetii, from the Mediterranean Sea (490 ng.g

w.wt). Among fish, the Myctophid Notoscopelus kroeyeri from the Atlantic displayed the highest Hg level (105 ± 80 ng.g

w.wt.). However, no Myctophids from the Mediterranean area could be sampled.

Cd concentrations in cephalopods displayed high intra- and inter-specific variability, and especially for the Mediterranean Sea where samples had the lowest variability in their mean length (Table 6). For example, the squid Loligo vulgaris was characterised by the lowest Cd levels in both Atlantic and Mediterranean Sea (132 ± 54 and 99 ± 111 ng.g

w.wt., respectively) but also by the highest CV (41 and 112%, respectively). In the Atlantic, Cd levels in cephalopods could be described following this pattern: Teuthowenia megalops (Cranchids) > Histioteuthis reversa (Histioteuthids) > I. coindetii (Ommastrephids) > L.

vulgaris (Loliginids).

4. Discussion

Metal concentrations displayed high variability in storage tissues of striped and bottlenose dolphins from the Mediterranean and Atlantic areas. The extreme CV values obtained in our study (i.e. 94 to 221%) have previously been reported in other marine mammal species for these toxic elements (e.g. Caurant et al., 1994; Bustamante et al., 2004). This underlines the fact that metal levels in upper-level predators are likely to be influenced by numerous factors, such as age, gender, body condition, geographical location and/or dietary preferences (Aguilar et al., 1999).

Concerning the influence of gender, no effect was detected in striped dolphins stranded along the Atlantic coast for either hepatic Hg or renal Cd concentrations (Table 4). This result is consistent with those obtained for striped dolphins from the Pacific Ocean (Honda et al., 1983). Some differences of Cd concentrations between genders have been reported for other marine mammals, e.g. long-finned pilot whales Globicephala melas and grey seals Halichoerus grypus, and were attributed to their sexual dimorphism (Caurant et al., 1994;

Bustamante et al., 2004). Although male striped dolphins from the Atlantic Ocean are slightly

bigger than females (Di-Méglio et al., 1996), this sexual dimorphism might not be sufficient

to induce differences in Cd levels between genders. This also suggests that the transfer of Cd

to offspring during pregnancy and lactation is not an important route of excretion for female

striped dolphins.

4.1. Mercury

Age was the major factor affecting hepatic Hg concentrations in bottlenose and striped dolphins from the Atlantic and Mediterranean waters (Table 3). Such an increase of Hg concentrations with age has previously been demonstrated in several cetacean species (e.g.

Honda et al., 1983; Paludan-Müller et al., 1993; Caurant et al., 1994; Monaci et al., 1998).

Comparisons between the two studied species do not reveal any difference in Hg accumulation in the Atlantic (Table 5). Such similar Hg accumulation trends between bottlenose (i.e. mainly fish-eaters in this area: Spitz, 2004) and striped dolphins (i.e. mixed item-eaters: Pusineri et al., in press; Spitz et al., submitted) are consistent with the high bioavailability of Hg in both fish and cephalopods (Bloom, 1992; Bustamante et al., submitted). However, geographical comparisons highlighted two different patterns: 1) bottlenose dolphins from the Mediterranean Sea displayed higher Hg concentrations than Atlantic ones whereas 2) Mediterranean and Atlantic striped dolphins exhibited similar Hg concentrations at a given age (Fig. 2a, Fig. 3a, Table 3).

Firstly, the Hg accumulation in Mediterranean bottlenose dolphins compared to Atlantic

animals resulted in mean Hg levels about 5 times higher in Mediterranean bottlenose dolphins

than Atlantic animals (i.e. 204 ± 121 vs 38 ± 41 µg.g

w.wt., respectively). Mediterranean

prey displayed higher Hg levels than Atlantic prey (Table 6). Thus, higher Hg levels in

Mediterranean prey and Mediterranean bottlenose dolphins were in agreement with the Hg

enrichment in Mediterranean food webs, due to higher methylation rates of Hg in the anoxic

layer waters of this area (Bacci, 1989; Cossa et al., 1997).

Surprisingly, and conversely to bottlenose dolphins, no difference in Hg bioaccumulation was established between Mediterranean and Atlantic striped dolphins. The highest Hg concentration (1033 µg.g

w.wt.) was encountered in a putrefied 27 year-old Mediterranean dolphin. Such an extreme elevated level has been previously reported for several striped dolphins from the Mediterranean Sea (see Table 7), but without taking into account their age or body condition. In their previous report on Hg levels in striped dolphins from the Atlantic and the Mediterranean Sea, André et al. (1991) used mostly mature animals, which had reached their asymptotic length. As the life-span of striped dolphins might reach 40 years (Miyasaki, 1981; Honda et al., 1983), these individuals could also have been from about 10 to 40 years old, which makes it difficult to establish geographical comparisons for these individuals. Therefore, the previous report of higher Hg levels in Mediterranean striped dolphins compared to Atlantic ones (André et al., 1991) can not be assessed. This underlines the fact that relationships of metal concentrations with body length is not appropriate for geographical comparisons, especially when growth curves are clearly different (Di-Méglio et al., 1996). However, if the environmental chemistry of water mass was considered alone, the Mediterranean striped dolphins would display higher Hg concentrations than the Atlantic ones. Hence, feeding and habitat preferences need to be taken into account.

In the Bay of Biscay, striped dolphins are mainly found in oceanic waters, where they

primarily feed upon on mesopelagic fish and cephalopods (Pusineri et al., in press). Some

intrusions over the continental shelf could also occur (Forcada et al., 1990; Kiszka et al., in

press), as underlined by the simultaneous occurrence of neritic and oceanic species in

stomachs of stranded animals (Spitz et al., submitted). In contrast, in north-western

Mediterranean waters, striped dolphins would occur equally over the continental shelf, the

shelf-edge and in deeper waters (Gannier, 1995). The diet of stranded Mediterranean striped

dolphins is mainly based on cephalopods and demersal fish from the upper shelf, at comparable proportions in terms of reconstructed biomass (Astruc et al., in press). Regarding Hg levels in the Atlantic prey, Myctophid fish were the prey with the highest concentrations (Table 6). Indeed, these mesopelagic fish are well-known to exhibit particularly high Hg levels (Monteiro et al., 1996; Thompson et al., 1998). No Mediterranean Myctophids could be sampled in this study but these mesopelagic fish could be expected to also present elevated mercury levels, probably higher than those from the Atlantic as a result of the Hg enrichment in this area (Bacci 1989). Our results concerning Hg concentrations in the liver of the striped dolphins strongly suggest that they do not feed on mesopelagic prey on a long time-scale in the Mediterranean waters in contrast to those from the Atlantic. Thus, different feeding behaviour could explain similar Hg tissue concentrations because mesopelagic prey species in the Atlantic display similar Hg levels to demersal prey in the Mediterranean, leading to similar Hg exposure in both areas.

4.2. Cadmium

Contrary to Hg, different patterns of Cd bioaccumulation occurred between the two studied

species. Indeed, despite the limited number of bottlenose dolphins sampled (see Table 1),

comparisons between the two species in the Atlantic revealed far lower Cd levels at a given

age in bottlenose dolphins than in striped dolphins (Table 5). Some piscivorous marine

mammals such as harbour porpoise (Phocoena phocoena), belugas (Delphinapterus leucas),

grey seals (H. grypus) and bottlenose dolphins from other areas also accumulate low levels of

Cd (e.g. Falconer et al., 1983; Wagemann et al., 1996; Szefer et al., 2002; Bustamante et al.,

2004; Decataldo et al., 2004). The low Cd levels encountered in bottlenose dolphins would be

induced by a diet dominated by fish (Spitz, 2004), which contain low Cd levels (Amiart-

Triquet et al., 1983; Bustamante, 1998; Lahaye et al., in press). In contrast, regarding elevated Cd levels in striped dolphins, high Cd concentrations have also been reported in the tissues of teuthophageous species (e.g. Caurant et al., 1994; Holsbeek et al., 1999; Bustamante et al., 2003). As feeding on cephalopods constitutes a major source of Cd for small cetaceans (Bustamante et al., 1998; 2002), the elevated Cd concentrations in striped dolphins well- reflected their important squid consumption in the Atlantic area (Pusineri et al., in press; Spitz et al., submitted).

No difference in Cd accumulation was found in bottlenose dolphins from the Atlantic and Mediterranean areas (Table 3). However, the 3 individuals from Arcachon Bay displayed particularly low Cd concentrations compared to the individuals stranded along the Atlantic coast (Fig. 1b). Consequently, the low Cd levels encountered in the resident bottlenose dolphins from Arcachon Bay may be the result of a specialised diet resulting from particular prey availability in this restricted area. Most of the fish encountered are coastal species rather than demersal ones and the main source of cephalopods in this bay is common cuttlefish (Sepia officinalis). Indeed, Arcachon Bay is a mating area for this species, which die after the spawning, adults therefore being present in this area only during a very limited period of the year (Poulard and Léauté, 2002). Consequently, the Cd levels obtained in the bottlenose dolphins from Arcachon Bay were clearly due to the even more limited occurrence of cephalopods in their feeding regime compared to the other individuals from the Atlantic area, for which stomachs generally contain about 10% of cephalopods in term of reconstructed biomass (Spitz, 2004).

In addition, Cd accumulated significantly with age in bottlenose dolphins which was not the

case for striped dolphins (Table 3). Like bottlenose dolphins, harbour porpoises and belugas

would exhibit increasing concentration of renal Cd during their life (Falconer et al., 1983;

Wagemann et al., 1996; Szefer et al. 2002). Concerning striped dolphins, the apparent increase of Cd in the kidneys until 2 years old, and then followed by a plateau (Fig. 3b), can not be statistically confirmed with our sampling. Indeed, when looking at calves of 2 years- old and less, renal Cd concentrations were more influenced by the geographical area than the age (Table 3). Because it was opportunistic, our sampling is however limited to a few individuals for the Mediterranean area (see Table 1). Our results fall within the range of Cd concentrations reported in previous studies in the Mediterranean Sea but such a comparison with previous studies also showed that immature Mediterranean striped dolphins from our study were smaller and also younger than those from other studies (see Table 7). The particularly low Cd levels encountered in Mediterranean calves compared to Atlantic calves may also be the consequence of the sampling characteristics of this study. Indeed there is no significant difference when interaction between age and area is considered (Table 3). In addition, the proportion of cephalopods in the diet did not seem to differ between Atlantic and Mediterranean striped dolphins, as revealed by stomach content analyses (Astruc et al., in press; Pusineri et al., in press; Spitz et al., submitted). Given that Mediterranean cephalopods displayed similar or even higher Cd levels compared to Atlantic cephalopods (Table 6), the similar Cd concentrations measured in the kidneys of two Mediterranean striped dolphins compared to Atlantic ones may reflect a similar Cd impregnation state for these two populations.

A larger sample size is also required to better understand Cd accumulation with age in

Atlantic and Mediterranean striped dolphins. Indeed, a logarithmic pattern of Cd

accumulation was previously described for Pacific striped dolphins (Honda et al., 1983), as

well as in long-finned pilot whales G. melas (Caurant et al., 1994), and arctic narwhals

Monodon monoceros (Dietz et al., 2004), which consume a large proportion of squids. A rapid increase of Cd concentrations in calves could be explained by transfer of the metal through milk during the suckling period (Honda et al., 1983) as well as a higher absorption efficiency generally exhibited by young mammals (Underwood, 1977). Hence, Cd concentrations increased rapidly in calves to levels closed to the threshold likely to induce toxic effects (Dietz et al., 1998) (i.e. up to 40 µg.g

w.wt., see Fig. 3b). The fact that these concentrations would be maintained constant for the rest of the striped dolphin’s life (Fig. 3b) suggests an efficient adaptation of accumulation processes in response to high exposure to Cd through the consumption of cephalopods.

5. Conclusions

The present study underlines the importance of considering age when making geographical

comparisons of toxic element accumulation in upper-level predators. Hence, taking into

account age as a covariate has permitted us to control for biometric differences in the two

populations of striped dolphins considered in this study. Thus, the similar Hg levels obtained

in Atlantic and Mediterranean striped dolphins highlighted the influence of feeding behaviour

on Hg concentrations and suggested that striped dolphins feed in mesopelagic waters of the

Atlantic whereas striped dolphins from the Mediterranean Sea feed on other species over

shallower waters. Further investigations on feeding behaviour of striped dolphin and

quantitative estimation of exposure to metals through the diet are needed, and especially for

other areas of the Mediterranean Sea. Moreover, Cd concentrations in the kidneys would

well-reflect the occurrence of cephalopods in the diet on a long time-scale, as underlined by

the particular Cd impregnation state of animals that have inhabited Arcachon Bay throughout

their life. Concerning striped dolphins, the similar Cd impregnation state in Mediterranean

and Atlantic populations obtained when taking age into account will have to be re- investigated in the future with a larger sample size. Overall, our results suggest that metal measurements could constitute efficient additional tracers to better understand long-term feeding preferences of these pelagic upper-level predators.

Acknowledgements

We are very grateful to all the participants of the French national stranding network (RNE) for the collection of dolphin samples and to IFREMER for facilitating the prey sampling.

Thanks to V. Loizeau and C. Melon for collecting Mediterranean prey, and to C. Churlaud from the Centre Commun d’Analyses from La Rochelle for technical assistance. We also wish to thank A. Guerra and A. González of the Instituto de Investigaciones Marinas of Vigo for the species determination of oceanic squids and J.J. Boubert of the Réserve Naturelle du Banc d’Arguin for providing information about the bottlenose dolphins from the Arcachon Bay.

This work was funded by the European program BIOCET (EC: EVK3-CT-2000-00027) and through a research grant from the Conseil Régional de Poitou-Charentes of V.L.

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Figure captions

Figure 1. Studied areas in the north-eastern Atlantic waters and the north-western Mediteranean Sea. The continental shelf is represented in grey.

Figure 2. Relationships between age and (a) hepatic Hg and (b) renal Cd concentrations (µ g.g

-1

w.wt.) in bottlenose dolphins stranded along the French Atlantic (white squares) and Mediterranean (black squares) coasts. Crosses represent the 3 individuals from Arcachon Bay.

Figure 3. Relationships between age and (a) hepatic Hg and (b) renal Cd concentrations (µ g.g

-1

w.wt.) in striped dolphins stranded along the French Atlantic (white squares) and

Mediterranean (black squares) coasts.

Figure 1

Arcachon Bay Cap Breton trough

Gulf of L ion

Figure 2

0.01 0.1 1 10

0 5 10 15 20 25 30 35

Age R en a l [C d ] (µ g .g

w .w t. )

1 10 100 1000

0 5 10 15 20 25 30 35

Age

H ep a ti c [H g ] (µ g .g

w .w t. )

Figure 3

0.01 0.1 1 10 100

0 5 10 15 20 25 30

Age R en a l [C d ] (µ g .g

w .w t. )

1 10 100 1000 10000

0 5 10 15 20 25 30

Age

H ep a ti c [H g ] (µ g .g

w .w t. )

Tables

Table 1. Characteristics of the striped (n = 40) and bottlenose dolphins (n = 15) sampled between 1999 and 2004 along the Atlantic and Mediterranean French coasts.

* 3 individuals were originated from the resident group of Arcachon Bay

Species

Sampling area liver kidney < 2 y. old older

nd

Stenella coeruleoalba

Bay of Biscay 30 30 12 18 17 13 -

Mediterranean Sea 10 6 6 4 6 3 1

Tursiops truncatus

Bay of Biscay 10* 9* 3 7 5 5 -

Mediterranean Sea 5 3 1 4 1 4 -

Tissue Age category Gender

Table 2. Toxic element levels (mean ± SD; µ g.g

d.wt.) in standard reference materials (with 6 replicates).

certified value

observed value

certified value

observed value

certified value

observed value

Hg 0.27 ± 0.06 0.28 ± 0.003 4.64 ± 0.26 nd 3.37 ± 0.14 3.27 ± 0.07 Cd 26.7 ± 0.6 26.2 ± 0.2 0.046 ± 0.008 0.046 ± 0.001 19.4 ± 0.6 19.2 ± 0.4 Metals

Standard reference materials (NRCC)

TORT-2 DORM-2 DOLT-3

Table 3. Influence of age and area on metal levels in small cetaceans stranded along the French Mediterranean and Atlantic coasts, determined by ANCOVA. Influence of the studied parameters is given with Fisher’s F and its associated p-value (in brackets).

* ANCOVA on renal Cd concentrations refers to young striped dolphins (≤ 2 years old), for which Cd accumulation with age was linear.

T. truncatus

hepatic Hg 8 0.900

1.286 (p = 0.290)

renal Cd 5 0.978

5.198 (p = 0.072) 2.105 (p = 0.207)

hepatic Hg 36 0.820

0.116 (p = 0.735) 0.160 (p = 0.691) renal Cd* 12 0.767 0.237 (p = 0.635)

0.115 (p = 0.741)

Species

ddl

Studied parameters

Age Area Age x Area

Metal R²

Table 4. Influence of age and gender on metal levels in Atlantic striped dolphins. Influence of the studied parameters is given with Fisher’s F and its associated p-value (in brackets).

hepatic Hg ANCOVA 26 0.778

0.442 (p = 0.512) 0.229 (p = 0.367)

renal Cd ANOVA 28 0.002 0.051 (p = 0.822) 0.435 (p = 0.515) nd

Metal Statistical ddl

test R²

Studied parameters

Age Gender Age x Gender

Table 5. Variations of metal concentrations between species at the Atlantic scale, determined using ANCOVA. Influence of the studied parameters is given with Fisher’s F and its associated p-value (in brackets).

hepatic Hg 33 0.790

0.075 (p = 0.786) 0.084 (p = 0.774) renal Cd 32 0.599

Metal ddl

Studied parameters

Age Species Age x species

R²

Table 6. Toxic element levels (ng.g

w.wt.) in prey from the Atlantic and the Mediterranean Sea.

TAXON Length (mm)* [Hg] (ng.g^-1 w.wt) [Cd] (ng.g^-1 w.wt)

Family

Species Area n % H mean ± sd range CV% mean ± sd range CV% mean ± sd range CV%

FISH Clupeidae

Sardina pilchardius Atl. 6 61 154 ± 4 148 - 159 2.6 23 ± 7 19 - 34 26 nd nd nd

Med. 6 61 145 ± 6 137 - 150 3.8 94 ± 22 67 - 127 24 nd nd nd

Engraulidae

Engraulis encrasicolus Atl. 6 70 118 ± 4 112 - 122 3 28 ± 20 26 - 32 7.2 nd nd nd

Med. 6 73 109 ± 4 104 - 113 4.1 50 ± 9 43 - 61 17 nd nd nd

Gadidae

Micromesistius poutassou Atl. 3 75 141 ± 2 139 - 144 1.7 16 ± 1 16 - 17 5.2 nd nd nd

Med. 3 75 135 ± 4 131 - 138 2.7 21 ± 2 20 - 23 8.5 nd nd nd

Merluccidae

Merluccius merluccius Atl. 3 79 257 ± 42 225 - 305 16 33 ± 14 18 - 46 42 nd nd nd

Med. 3 79 222 ± 6 215 - 226 2.7 45 ± 12 34 - 58 26 nd nd nd

Myctophidae

Notoscopelus kroeyeri Atl. 6 70 93 ± 23 70 - 125 23 105 ± 80 29 - 210 77 nd nd nd

CEPHALOPOD Cranchidae

Teuthowenia megalops Atl. 1 83 181 34 1320

Histioteuthidae

Histioteuthis reversa Atl. 5 85 40 ± 24 20 - 80 59 15 ± 5 10 - 24 34 383 ± 196 148 - 665 21

Loliginidae

Loligo vulgaris Atl. 26 77 148 ± 50 73 - 256 34 56 ± 22 24 - 97 40 132 ± 54 58 - 238 41

Med. 12 74 173 ± 54 120 - 265 31 146 ± 66 68 - 250 45 99 ± 111 20 - 434 112 Ommastrephidae

Illex coindetii Atl. 22 79 130 ± 54 73 - 272 42 41 ± 17 14 - 69 41 290 ± 94 150 - 520 32

Med. 6 67 138 ± 19 118 - 161 14 277 ± 150 110 - 490 54 697 ± 391 380 - 1290 56

With: %H, percent of humidity; nd not determined. * Length refers to standard length for fish and mantle length for cephalopod.

Table 7. Hepatic Hg and renal Cd levels (µ g.g

w.wt.) in striped dolphins obtained in previous works.

With:

d.wt. converted in w.wt. on the basis of the d.wt. : w.wt. ratio obtained in this study;

and

refers to median. NB: maturity has been assumed to be “immature” when body length was < 175 cm for Mediterranean animals, and < 200 cm for other animals.

Maturity n mean ± sd range n mean ± sd range

Bay of Biscay oceanic waters

immature 153 ± 27 13 - - 15 17.3 ± 12.7 0.026 - 43.8 Das et al., 2000b

mature 214 ± 7 5 - - 8 27.9 ± 11.2 10.7 - 45.8

stranding

immature 165 ± 17 17 6.5 ± 6 1.2 - 24.1 17 12.9 ± 10.8 0.29 - 40.2 this study

mature 219 ± 12 13 138 ± 92 6.4 - 317 13 10.6 ± 9.0 2.1 - 30.8

immature 180 ± 19 3 23.7 ± 20.4 1.2 - 41 - - André et al. 1991

mature 211 ± 11 5 68.4 ± 18.3 46 - 87 - -

mature 185 2 10.2^a 9.8 - 10.6^a 31.3^a 30.4 - 32.2^a Holsbeek et al. 1998

Mediterranean Sea NW, France

immature 105 ± 21 6 4.4 ± 2.5 1.1 - 8.2 4 0.095 ± 0.07 0.02 - 0.18 this study

mature 195 ± 8 4 373 ± 454 29.5 - 1033 2 12.6 4.9 - 20.3

immature 135 ± 30 5 15.4 ± 20.4 1.2 - 49 - - André et al. 1991

mature 198 ±11 20 429 ± 352 4.7 - 1544 - -

immature 173 2 32.3^a 19 - 45.5^a - - Augier et al. 1993

mature 196 ± 8 9 159 ± 83^a 48.4 - 290^a - -

immature 137 ± 36 6 42.9 ± 32.9 3.4 - 81 - - Capelli et al. 2000

mature 196 ± 15 9 194 ± 207 37.9 - 692 - -

not detailed - 46 166 ± 314^a,b - 39 6.3 ± 7.2^a,b - Monaci et al. 1998

NW, Spain

not detailed - 20 292 ± 234^a,b - 20 1.9 ± 1.5^a,b - Monaci et al. 1998

NW, Italy

not detailed - 19 90.8^a,b 3.5 - 1232^a 18 10.3^a,b 2.5 - 22.7^a Leonzio et al. 1992

Southern Italy

immature 107 1 1.96 - - - Cardellicchio et al. 2000

mature - 6 189 ± 29 156 - 217 6 7 ± 4.1 1.8 - 12.8

immature 139 ± 29 3 5.9 ± 5.6 2.3 - 12.3 - - Cardellicchio et al. 2002a

mature 198 ± 10 7 241 ± 98 107 - 375 - -

not detailed 180 ± 33 - - 10 6.4 ± 4.3 0.3 - 15.7 Cardellicchio et al. 2002b

immature 134 ± 36 3 9.6 ± 11.5 0.8 - 22.7 3 0.51 ± 0.85 0.01 - 1.5 Decataldo et al. 2004

mature 187 ± 13 6 179 ± 197 13 - 463 6 5.7 ± 4.3 1.5 - 12.6

not detailed - 30 277 ± 246 0.58 - 966 - - Storelli et al. 1998

Bristish Isles

mature - 3 8.9 ± 2.8 5.7 - 11 - - Law et al. 1992

Pacific

not detailed - 45 205 ± 138 1.7 - 485 30 24.8 ± 16.2 0.06 - 69.6 Honda et al. 1983

immature - 6 5.81 ± 2.76 - - - Itano et al. 1984

mature - 15 205 ± 102 - - -

Reference Length (cm)

Locality Hepatic Hg (µg.g^-1 w.wt.) Renal Cd (µg.g^-1 w.wt.)